Mitochondrial protein import machinery conveys stress signals to the cytosol and beyond.

Mitochondria hold diverse and pivotal roles in fundamental processes that govern cell survival, differentiation, and death, in addition to organismal growth, maintenance, and aging. The mitochondrial protein import system is a major contributor to mitochondrial biogenesis and lies at the crossroads between mitochondrial and cellular homeostasis. Recent findings highlight the mitochondrial protein import system as a signaling hub, receiving inputs from other cellular compartments and adjusting its function accordingly. Impairment of protein import, in a physiological, or disease context, elicits adaptive responses inside and outside mitochondria. In this review, we discuss recent developments, relevant to the mechanisms of mitochondrial protein import regulation, with a particular focus on quality control, proteostatic and metabolic cellular responses, triggered upon impairment of mitochondrial protein import.

MitoSNARE Assembly and Disassembly Factors Regulate Basal Autophagy and Aging in C. elegans.

SNARE proteins reside between opposing membranes and facilitate vesicle fusion, a physiological process ubiquitously required for secretion, endocytosis and autophagy. With age, neurosecretory SNARE activity drops and is pertinent to age-associated neurological disorders. Despite the importance of SNARE complex assembly and disassembly in membrane fusion, their diverse localization hinders the complete understanding of their function. Here, we revealed a subset of SNARE proteins, the syntaxin SYX-17, the synaptobrevins VAMP-7, SNB-6 and the tethering factor USO-1, to be either localized or in close proximity to mitochondria, in vivo. We term them mitoSNAREs and show that animals deficient in mitoSNAREs exhibit increased mitochondria mass and accumulation of autophagosomes. The SNARE disassembly factor NSF-1 seems to be required for the effects of mitoSNARE depletion. Moreover, we find mitoSNAREs to be indispensable for normal aging in both neuronal and non-neuronal tissues. Overall, we uncover a previously unrecognized subset of SNAREs that localize to mitochondria and propose a role of mitoSNARE assembly and disassembly factors in basal autophagy regulation and aging.

Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans.

Impaired mitochondrial maintenance in disparate cell types is a shared hallmark of many human pathologies and ageing. How mitochondrial biogenesis coordinates with the removal of damaged or superfluous mitochondria to maintain cellular homeostasis is not well understood. Here we show that mitophagy, a selective type of autophagy targeting mitochondria for degradation, interfaces with mitochondrial biogenesis to regulate mitochondrial content and longevity in Caenorhabditis elegans. We find that DCT-1 is a key mediator of mitophagy and longevity assurance under conditions of stress in C. elegans. Impairment of mitophagy compromises stress resistance and triggers mitochondrial retrograde signalling through the SKN-1 transcription factor that regulates both mitochondrial biogenesis genes and mitophagy by enhancing DCT-1 expression. Our findings reveal a homeostatic feedback loop that integrates metabolic signals to coordinate the biogenesis and turnover of mitochondria. Uncoupling of these two processes during ageing contributes to overproliferation of damaged mitochondria and decline of cellular function.

Mitochondria, autophagy and age-associated neurodegenerative diseases: New insights into a complex interplay.

Mitochondria represent the major bioenergetic hub coordinating cellular and organismal homeostasis. The underlying causes of many pathologies tormenting humans converge on impaired mitochondrial maintenance. Mitochondria-specific autophagy (mitophagy), a cellular catabolic process targeting mitochondria, holds a prominent role in mitochondrial quality control. In addition to core autophagic machinery components, mitophagy exploits a variety of molecules that identify damaged or superfluous mitochondria and mediate their elimination. Signaling pathways integrating environmental and genetic stimuli interact with key mitophagy effectors to activate cellular stress response mechanisms, ultimately modulating health and lifespan. Here, we review the signaling cascades and molecular mechanisms that govern the process of mitophagy and discuss their involvement in ageing and neurodegeneration. This article is part of a Special Issue entitled: Mitochondrial Dysfunction in Aging.

Molecular recognition and substrate mimicry drive the electron-transfer process between MIA40 and ALR.

Oxidative protein folding in the mitochondrial intermembrane space requires the transfer of a disulfide bond from MIA40 to the substrate. During this process MIA40 is reduced and regenerated to a functional state through the interaction with the flavin-dependent sulfhydryl oxidase ALR. Here we present the mechanistic basis of ALR-MIA40 interaction at atomic resolution by biochemical and structural analyses of the mitochondrial ALR isoform and its covalent mixed disulfide intermediate with MIA40. This ALR isoform contains a folded FAD-binding domain at the C-terminus and an unstructured, flexible N-terminal domain, weakly and transiently interacting one with the other. A specific region of the N-terminal domain guides the interaction with the MIA40 substrate binding cleft (mimicking the interaction of the substrate itself), without being involved in the import of ALR. The hydrophobicity-driven binding of this region ensures precise protein-protein recognition needed for an efficient electron transfer process.

Assessing locomotory rate in response to food for the identification of neuronal and muscular defects in C. elegans.

C. elegans is a bacteria-eating soil-dwelling nematode. Typical cultivation of laboratory-reared populations occurs on bacteria-covered solid media, where they move along with sinusoidal undulations. Nematodes decelerate when they encounter food. Dopaminergic and serotonergic neurotransmission regulate this behavior. Here, we describe the procedure for determining food-dependent locomotion rate of fed and fasting nematodes. We detail steps for assay plate preparation, C. elegans synchronization, and assessment of locomotion. The behaviors we describe provide information regarding the animal's physiological neuronal and muscular function. For complete details on the use and execution of this protocol, please refer to Petratou et al. (2023)1 and Sawin et al. (2000).2.

Heat shock and thermotolerance in Caenorhabditis elegans: An overview of laboratory techniques.

The soil nematode worm Caenorhabditis elegans is a simple and well-established model for the study of many biological processes. Heat shock and thermotolerance assays have been developed for this nematode, and have been used to decipher the molecular relationships between thermal stress and aging, among others. Nevertheless, a systematic and methodological comparison of the different approaches and tools utilized is lacking in the literature. Here, we aim to provide a comprehensive summary of the most commonly used strategies for carrying out heat shock and thermotolerance assays that have been reported, highlighting specific readouts and scientific questions that can be addressed. Furthermore, we offer examples of thermotolerance assays performed with wild type nematodes, that can serve as a gauge of the animal survival under diverse conditions of stress.

One-Carbon Metabolism: Pulling the Strings behind Aging and Neurodegeneration.

One-carbon metabolism (OCM) is a network of biochemical reactions delivering one-carbon units to various biosynthetic pathways. The folate cycle and methionine cycle are the two key modules of this network that regulate purine and thymidine synthesis, amino acid homeostasis, and epigenetic mechanisms. Intersection with the transsulfuration pathway supports glutathione production and regulation of the cellular redox state. Dietary intake of micronutrients, such as folates and amino acids, directly contributes to OCM, thereby adapting the cellular metabolic state to environmental inputs. The contribution of OCM to cellular proliferation during development and in adult proliferative tissues is well established. Nevertheless, accumulating evidence reveals the pivotal role of OCM in cellular homeostasis of non-proliferative tissues and in coordination of signaling cascades that regulate energy homeostasis and longevity. In this review, we summarize the current knowledge on OCM and related pathways and discuss how this metabolic network may impact longevity and neurodegeneration across species.

Mitochondrial protein import determines lifespan through metabolic reprogramming and de novo serine biosynthesis.

Sustained mitochondrial fitness relies on coordinated biogenesis and clearance. Both processes are regulated by constant targeting of proteins into the organelle. Thus, mitochondrial protein import sets the pace for mitochondrial abundance and function. However, our understanding of mitochondrial protein translocation as a regulator of longevity remains enigmatic. Here, we targeted the main protein import translocases and assessed their contribution to mitochondrial abundance and organismal physiology. We find that reduction in cellular mitochondrial load through mitochondrial protein import system suppression, referred to as MitoMISS, elicits a distinct longevity paradigm. We show that MitoMISS triggers the mitochondrial unfolded protein response, orchestrating an adaptive reprogramming of metabolism. Glycolysis and de novo serine biosynthesis are causatively linked to longevity, whilst mitochondrial chaperone induction is dispensable for lifespan extension. Our findings extent the pro-longevity role of UPRmt and provide insight, relevant to the metabolic alterations that promote or undermine survival and longevity.

Mitophagy Dynamics in Caenorhabditis elegans.

Mitochondrial selective autophagy (mitophagy) is a critical cellular process for mitochondrial homeostasis and survival both under basal and stress conditions. Distinct cell types display different requirements for mitochondrial turnover depending on their metabolic status, differentiation state, and environmental cues. This points to the necessity of developing novel tools for real-time, tissue-specific assessment of mitophagy. Caenorhabditis elegans is an invaluable model organism for this kind of analysis providing a platform for simultaneous monitoring of mitophagy in vivo in different tissues and cell types, during development, stress conditions, and/or throughout life span. In this chapter we describe three versatile, noninvasive methods, developed for monitoring in vivo early and late mitophagic events in body wall muscles and neuronal cells of C. elegans. These procedures can be readily used and/or provide insights into the generation of novel imaging methods to investigate further the role of mitophagy at the organismal level under normal and pathological conditions.

Mechanisms of mitophagy in cellular homeostasis, physiology and pathology.

Mitophagy is an evolutionarily conserved cellular process to remove dysfunctional or superfluous mitochondria, thus fine-tuning mitochondrial number and preserving energy metabolism. In this Review, we survey recent advances towards elucidating the molecular mechanisms that mediate mitochondrial elimination and the signalling pathways that govern mitophagy. We consider the contributions of mitophagy in physiological and pathological contexts and discuss emerging findings, highlighting the potential value of mitophagy modulation in therapeutic intervention.

Mitophagy: In sickness and in health.

Mitophagy is a conserved, mitochondria-specific autophagic clearance process. We recently discovered an intricate regulatory network that balances mitophagy with mitochondrial biogenesis. Proper coordination of these opposing processes is important for stress resistance and longevity. Nodal regulatory factors that contribute to mitochondrial homeostasis have also been linked to carcinogenesis, highlighting mitophagy as a potential target for therapeutic interventions against cancer.

Differential Protein Distribution between the Nucleus and Mitochondria: Implications in Aging.

The coordination of nuclear and mitochondrial genomes plays a pivotal role in maintenance of mitochondrial biogenesis and functionality during stress and aging. Environmental and cellular inputs signal to nucleus and/or mitochondria to trigger interorganellar compensatory responses. Loss of this tightly orchestrated coordination results in loss of cellular homeostasis and underlies various pathologies and age-related diseases. Several signaling cascades that govern interorganellar communication have been revealed up to now, and have been classified as part of the anterograde (nucleus to mitochondria) or retrograde (mitochondrial to nucleus) response. Many of these molecular pathways rely on the dual distribution of nuclear or mitochondrial components under basal or stress conditions. These dually localized components usually engage in specific tasks in their primary organelle of function, whilst upon cellular stimuli, they appear in the other organelle where they engage in the same or a different task, triggering a compensatory stress response. In this review, we focus on protein factors distributed between the nucleus and mitochondria and activated to exert their functions upon basal or stress conditions. We further discuss implications of bi-organellar targeting in the context of aging.

Interfacing mitochondrial biogenesis and elimination to enhance host pathogen defense and longevity.

Mitochondria are highly dynamic and semi-autonomous organelles, essential for many fundamental cellular processes, including energy production, metabolite synthesis and calcium homeostasis, among others. Alterations in mitochondrial activity not only influence individual cell function but also, through non-cell autonomous mechanisms, whole body metabolism, healthspan and lifespan. Energy homeostasis is orchestrated by the complex interplay between mitochondrial biogenesis and mitochondria-selective autophagy (mitophagy). However, the cellular and molecular pathways that coordinate these 2 opposing processes remained obscure. In our recent study, we demonstrate that DCT-1, the Caenorhabditis elegans homolog of the mammalian BNIP3 and BNIP3L/NIX, is a key mediator of mitophagy, and functions in the same genetic pathway with PINK-1 and PDR-1 (the nematode homologs of PINK1 and Parkin respectively) to promote longevity and prevent cell damage under stress conditions. Interestingly, accumulation of damaged mitochondria activates SKN-1 (SKiNhead-1), the nematode homolog of NRF2, which in turn initiates a compensatory retrograde signaling response that impinges on both mitochondrial biogenesis and removal. In this commentary, we discuss the implications of these new findings in the context of innate immunity and aging. Unraveling the regulatory network that governs the crosstalk between mitochondrial biogenesis and mitophagy will enhance our understanding of the molecular mechanisms that link aberrant energy metabolism to aging and disease.

Coupling mitogenesis and mitophagy for longevity.

Maintenance of mitochondrial function and energy homeostasis requires both generation of newly synthesized and elimination of dysfunctional mitochondria. Impaired mitochondrial function and excessive mitochondrial content are major characteristics of aging and several human pathophysiological conditions, highlighting the pivotal role of the coordination between mitochondrial biogenesis and mitophagy. However, the cellular and molecular underpinnings of mitochondrial mass homeostasis remain obscure. In our recent study, we demonstrate that DCT-1, the Caenorhabditis elegans homolog of mammalian BNIP3 and BNIP3L/NIX, is a key mediator of mitophagy promoting longevity under stress. DCT-1 acts downstream of the PINK-1-PDR-1/Parkin pathway and is ubiquitinated upon mitophagy-inducing conditions to mediate the removal of damaged mitochondria. Accumulation of damaged mitochondria triggers SKN-1 activation, which initiates a bipartite retrograde signaling pathway stimulating the coordinated induction of both mitochondrial biogenesis and mitophagy genes. Taken together, our results unravel a homeostatic feedback loop that allows cells to adjust their mitochondrial population in response to environmental and intracellular cues. Age-dependent decline of mitophagy both inhibits removal of dysfunctional or superfluous mitochondria and impairs mitochondrial biogenesis resulting in progressive mitochondrial accretion and consequently, deterioration of cell function.

Assessing aging and senescent decline in Caenorhabditis elegans: cohort survival analysis.

The nematode Caenorhabditis elegans provides a versatile and expedient platform for the genetic and molecular dissection of mechanisms underlying senescent decline and aging. Indeed, pioneering studies in this organism revealed the first genes and pathways directly influencing lifespan. In this chapter, we present routine, mainstream methods, developed for monitoring aging and senescent decline in C. elegans. These procedures allow the assessment of lifespan parameters on solid growth media that are typically used for rearing nematodes.

High-throughput and longitudinal analysis of aging and senescent decline in Caenorhabditis elegans.

Caenorhabditis elegans is becoming a multipurpose tool for genetic and chemical compound screening approaches aiming to identify and target the molecular mechanisms underlying senescent decline, aging, and associated pathologies. In this chapter, we describe specialized methods that have been developed to facilitate such screening strategies using C. elegans. The first section provides detailed procedures for the assessment of lifespan parameters on liquid growth media that are typically used for rearing nematodes. In the second section, we consider methodologies optimized for high-throughput survival analysis, applicable to large-scale chemical compound or genetic screening ventures. Finally, we discuss recently developed microfluidics tools for the noninvasive monitoring of behavioral and physiological traits in longitudinal studies of aging and senescent decline.

Oxidative stress and mitochondrial protein quality control in aging.

Mitochondrial protein quality control incorporates an elaborate network of chaperones and proteases that survey the organelle for misfolded or unfolded proteins and toxic aggregates. Repair of misfolded or aggregated protein and proteolytic removal of irreversibly damaged proteins are carried out by the mitochondrial protein quality control system. Initial maturation and folding of the nuclear or mitochondrial-encoded mitochondrial proteins are mediated by processing peptidases and chaperones that interact with the protein translocation machinery. Mitochondrial proteins are subjected to cumulative oxidative damage. Thus, impairment of quality control processes may cause mitochondrial dysfunction. Aging has been associated with a marked decline in the effectiveness of mitochondrial protein quality control. Here, we present an overview of the chaperones and proteases involved in the initial folding and maturation of new, incoming precursor molecules, and the subsequent repair and removal of oxidized aggregated proteins. In addition, we highlight the link between mitochondrial protein quality control mechanisms and the aging process. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

Autophagy and ageing: insights from invertebrate model organisms.

Ageing in diverse species ranging from yeast to humans is associated with the gradual, lifelong accumulation of molecular and cellular damage. Autophagy, a conserved lysosomal, self-destructive process involved in protein and organelle degradation, plays an essential role in both cellular and whole-animal homeostasis. Accumulating evidence now indicates that autophagic degradation declines with age and this gradual reduction of autophagy might have a causative role in the functional deterioration of biological systems during ageing. Indeed, loss of autophagy gene function significantly influences longevity. Moreover, genetic or pharmacological manipulations that extend lifespan in model organisms often activate autophagy. Interestingly, conserved signalling pathways and environmental factors that regulate ageing, such as the insulin/IGF-1 signalling pathway and oxidative stress response pathways converge on autophagy. In this article, we survey recent findings in invertebrates that contribute to advance our understanding of the molecular links between autophagy and the regulation of ageing. In addition, we consider related mechanisms in other organisms and discuss their similarities and idiosyncratic features in a comparative manner.

The N-terminal shuttle domain of Erv1 determines the affinity for Mia40 and mediates electron transfer to the catalytic Erv1 core in yeast mitochondria.

Erv1 and Mia40 constitute the two important components of the disulfide relay system that mediates oxidative protein folding in the mitochondrial intermembrane space. Mia40 is the import receptor that recognizes the substrates introducing disulfide bonds while it is reduced. A key function of Erv1 is to recycle Mia40 to its active oxidative state. Our aims here were to dissect the domain of Erv1 that mediates the protein-protein interaction with Mia40 and to investigate the interactions between the shuttle domain of Erv1 and its catalytic core and their relevance for the interaction with Mia40. We purified these domains separately as well as cysteine mutants in the shuttle and the active core domains. The noncovalent interaction of Mia40 with Erv1 was measured by isothermal titration calorimetry, whereas their covalent mixed disulfide intermediate was analyzed in reconstitution experiments in vitro and in organello. We established that the N-terminal shuttle domain of Erv1 is necessary and sufficient for interaction to occur. Furthermore, we provide direct evidence for the intramolecular electron transfer from the shuttle cysteine pair of Erv1 to the core domain. Finally, we reconstituted the system by adding in trans the N- and C- terminal domains of Erv1 together with its substrate Mia40.